Developmental Patterning as a Quantitative Trait: Genetic Modulation of the Hoxb6 Mutant Skeletal Phenotype

Abstract

The process of patterning along the anterior-posterior axis in vertebrates is highly conserved. The function of Hox genes in the axis patterning process is particularly well documented for bone development in the vertebral column and the limbs. We here show that Hoxb6, in skeletal elements at the cervico-thoracic junction, controls multiple independent aspects of skeletal pattern, implicating discrete developmental pathways as substrates for this transcription factor. In addition, we demonstrate that Hoxb6 function is subject to modulation by genetic factors. These results establish Hox-controlled skeletal pattern as a quantitative trait modulated by gene-gene interactions, and provide evidence that distinct modifiers influence the function of conserved developmental genes in fundamental patterning processes.

Fig 1. Hoxb6 mutants exhibit skeletal alterations and absence of the first pair of ribs.

Panel A: Rib cage of wildtype skeleton with 7 ribs attached to the sternum and 6 sternebrae. Panel B: Rib cage of mutant with 6 ribs attached to sternum and 5 sternebrae. Panel C: Cervico-thoracic region of wildtype newborn skeleton. Panel D: Cervico-thoracic region of Hoxb6^hd mutant newborn skeleton, the red arrow points to small ossified structures in place of the first pair of ribs. Panel E: E13.5 embryo heterozygous for the Hoxb6^hd mutant allele stained with Alcian Blue reveals normal cartilage anlagen. Panel F: E13.5 homozygous Hoxb6^hd mutant embryo with cartilage anlagen for the first rib absent (compare targets of black arrows). Panels G, H: Magnifications of Panels E and F, respectively. Panel I: Whole mount in situ hybridization for Hoxb6 in an embryo isolated at day E10.5. The anterior limit of Hoxb6 expression in somites is found within the caudal region of the somite that contributes to prevertebra 6 (red arrow). Panel J: In situ hybridization to a sagittal section from an embryo at E14.5 shows strong signal in spinal cord, and Hoxb6 expression is evident in the vertebral column (light blue arrow), and in the precursors to the sternum (yellow arrow).

Fig 2. Features of rib development and sternal articulation in Hoxb6^hd mutants.

Panel A: Newborn wildtype skeleton with outlines (white) of cartilaginous portions of the ribs and sternum. Panels B-K: Hoxb6^hd mutant skeletons from newborns (B-H) or adults (I-K). Panel B: Note the crossover of rib from the eighth vertebra and fusion with the rib from ninth vertebra, bifurcation of cartilage, and aberrant attachment of fused cartilage, and fusion of the first two sternebrae. Panels C-H: Different combinations in individuals of absent or rudimentary ribs, unilaterally (C-E) or bilaterally (F-H), defective formation of rib cartilage (D, E, G, H), crossovers (D, E, H), fusions (E, H), bifurcations (D, E, H), aberrant articulation to the sternum (C-H) and off-set sternal attachment of the ribs (H). All preparations contain the eighth and ninth vertebrae, except in Panel F, which represents the ninth vertebra. Panels I-K: X-rays of individual adult Hoxb6^hd mutants. Panel I: frontal view; note truncation of first rib unilaterally (red arrow). Panel J: lateral view, orange arrow points to crossover and defective sternal rib cartilage. Panel K: lateral view, dark yellow arrow points to crossover and unilateral absence of first rib, bright yellow arrow points to absence of spinous process on the ninth vertebra.

Fig 3. Phenotype expressivity in Hoxb6 mutants on different genetic backgrounds.

Panels in column A: Vertebral elements of wildtype newborn mouse skeleton. Arrows point to characteristic features: Vertebra C6: vertebral foramen and anterior tuberculum; C7: lateral extension of the vertebral body and absence of foramen; T1: articulation of rib capitulum to vertebral body; T2: dorsal cartilage extension (processus spinosus). Panels in column B: Homeotic transformations in a homozygous Hoxb6^hd mutant on mixed background. Arrows point to features found transposed to vertebrae at the next axial level, resembling anterior homeotic transformations of these skeletal elements. Panels in column C: Homeotic transformations in homozygous Hoxb6^hd mutant on C57BL/6 background. Homeotic transformations are more often found unilaterally; arrow points to rib cartilage associated with the transformed eighth vertebra.

Fig 4. Breeding scheme for C57BL/6-Hoxb6^hd congenic strain and backcross to 129S6/SvEv.

A male heterozygote for the Hoxb6^hd mutation was crossed to C57BL/6 wildtype females and male offspring were used for further backcrosses. For the backcross to 129S6/SvEv wildtype, a Hoxb6^hd homozygous male was used, and offspring from this cross (H₁) were intercrossed (H₁i) to generate mixed background animals homozygous for the Hoxb6 mutation. Out of 11 H₁i progeny, 3 had defective first rib development, indicating an effect of the 129S6/SvEv genetic background on phenotype manifestation. Further backcrosses used H₁ males heterozygous for the Hoxb6^hd mutation and wildtype 129S6/SvEv females. Intercrosses of H₃ animals (H₃i) yielded homozygous Hoxb6^hd mutants on predominantly 129S6/SvEv genetic background. To exclude a possible developmental disadvantage for the 25% homozygotes in a cross of Hoxb6^hd heterozygous parents, we set up crosses between H₃i generation animals in which the father was homozygous for the Hoxb6^hd mutation (H₃i-fh), thus increasing the yield of homozygous mutants to 50%, providing equal chance for intrauterine development. Of 38 mutant H₃i progeny, 33 exhibited the “missing rib” phenotype, confirming the influence of genetic background on phenotype manifestation in Hoxb6^hd mutants. Further backcrosses used homozygous mutant H₁i progeny bred to C57BL/6-Hoxb6^hd congenics (G₈i), which produced 42 K₂ progeny, of which 12 exhibited defective ribs; crosses of Hoxb6^hd homozygous mutant F₁ hybrids to H₃ Hoxb6^hd homozygous mutants produced 62 J₂ animals, of which 55 had defective ribs. Differences in incidence of the phenotype are statistically significant between all groups (p = or < 0.01), except for the J₂/H₃ comparison. These results provide evidence that genetic background controls rib development in Hoxb6^hd mutants in intermediate fashion.

Fig 5. Phenotype expressivity in Hoxb6 mutants is controlled by genetic background of the embryo.

Columns show the quantitative distribution of features by skeletal element and genetic background. Phenotype features were scored exactly as shown by orange arrows in Fig 3. Panel A: 6^th vertebra; Panel B: 7^th vertebra; Panel C: 8^th vertebra (normally first thoracic vertebra); Panel D: Articulation of ribs/ossifications to the 8^th vertebrae; Panel E: Proximal (vertebral) ribs; Panel F: Distal (sternal) ribs; Panel G: Articulation to sternum at level of 8^th vertebra (normally T1); Panel H: Articulation to sternum at level of 9^th vertebra (normally T2). For each experimental group, the fraction of animals with a given phenotype is plotted, and the total number of animals is given as a number in the respective columns of Panel C. No attempt was made to depict side of unilateral anomalies; as there was no preference, they were grouped together with increasing severity to the right of each column. Columns 1–8 correspond to the following groups: 1: Progeny from crosses of homozygous C57BL/6-Hoxb6^hd mutant males to heterozygous C57BL/6-Hoxb6^hd mutant females (G₈i, see Fig 4) maintained on corn-cob bedding (C57 cb). 2: Progeny from crosses of homozygous C57BL/6-Hoxb6^hd mutant males to heterozygous C57BL/6-Hoxb6^hd mutant females (G₈i) maintained on synthetic fiber bedding (C57 fb). 3: Progeny (F₁) from crosses of homozygous H₃ mutant males to homozygous C57BL/6-Hoxb6^hd females (C57m: maternal uterine environment is C57BL/6). 4: Progeny (F₁) from crosses of homozygous C57BL/6-Hox-6^hd mutant males to homozygous H₃ mutant females (129m: maternal uterine environment is predominantly 129Sv/Ev). 5: Progeny (H₃i) from brother x sister matings of H₃ generation animals on predominantly 129S6/SvEv background. 6: Progeny from crosses of homozygous H₃ mutant males to heterozygous H₃ mutant females (H₃i-fh; homozygous father) on predominantly 129S6/SvEv background. 7: C57BL/6-Hoxb6^hd heterozygous mutants (Het C57). 8: Heterozygous Hoxb6^hd mutants from H₃i intercrosses (Het 129). Statistical significance was established by 2-tailed Fisher’s exact test. For comparison of heterozygotes on the two different genetic backgrounds (Columns 7 and 8), p-values were not significant except for T1 status (p = 0.0095). For complete p-value matrices for all pairwise comparisons for columns 1–6 see Table 1.

Fig 6. Association of skeletal features in individual Hoxb6^hd mutants.

Regression analyses were performed using a simplified scoring scheme for each character: two points were assigned for bilateral abnormalities, one point for unilateral abnormalities and 0 for wildtype manifestation. Each animal is represented by a square. The correlation coefficients (R) were determined using Microsoft Excel. Significance for relationships was assessed by ANOVA and was smaller than p = 0.05 (adjusted p<0.003 with correction for multiple comparisons) only for data in Panels D-F, K, L (solid regression curves). The presence of association indicates that the cellular and molecular processes producing a given feature are likely developmentally linked. The absence of relationship between skeletal anomalies within individual animals indicates that the various aspects of the Hoxb6^hd phenotype develop independently from each other, possibly controlled by different underlying molecular mechanisms.

Fig 7. Influence of genetic background on risk for skeletal abnormalities in Hoxb6 mutants.

The fraction of animals with abnormalities (uni- and bilateral combined) for each group was determined and risk was calculated relative to C57BL/6-Hoxb6^hd homozygous mutants. Relative Risk increases with increasing contribution from the 129S6/SvEv genetic background; for some features, this increase is significant (p<0.05) in both chi-square and Fisher's exact test (p-values are shown). Confidence intervals (horizontal bars) were determined for the comparisons of relative risk estimate (small vertical bar) for Hoxb6 mutants on F1 hybrid and 129S6/SvEv genetic background, respectively, based upon the results in S2 Table. Relative risk for anomalies in Hoxb6^hd mutants on C57BL/6 background was set to 1 (green line), and risk estimates whose confidence intervals do NOT include the value 1 are highly significant as indicated by red color of the data points.